Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. Epoxide hydrolases from microbial sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which furnishes the corresponding vicinal diol and remaining non-hydrolyzed epoxide in nonracemic form, enantioconvergent processes are possible. These are highly attractive as they lead to the formation of a single enantiomeric diol from a racemic oxirane, see, e.g., Steinreiber (2001) Curr. Opin. Biotechnol. 12:552-558.
Microsomal epoxide hydrolases are biotransformation enzymes that catalyze the conversion of a broad array of xenobiotic epoxide substrates to more polar diol metabolites, see, e.g., Omiecinski (2000) Toxicol. Lett. 112-113:365-370. Microsomal epoxide hydrolases catalyze the addition of water to epoxides in a two-step reaction involving initial attack of an active site carboxylate on the oxirane to give an ester intermediate followed by hydrolysis of the ester. Soluble epoxide hydrolase play a role in the biosynthesis of inflammation mediators, see, e.g., Morisseau (1999) Proc. Natl. Acad. Sci. USA 96:8849-8854.
Chiral molecules, including alcohols, α-hydroxy acids and epoxides, are important for the synthesis of pharmaceuticals, agrochemicals, as well as many fine chemicals. A major challenge in modern organic chemistry is to generate such compounds in high yields, with high stereo- and regioselectivities. Enantiopure epoxides are versatile synthons for the synthesis of numerous pharmaceuticals, agrochemicals and other high value compounds.
Currently available methods have drawbacks that limit their use in industrial applications. In recent studies, epoxide hydrolases (hereinafter “EHs”) have shown promise as biocatalysts for the preparation of chiral epoxides and vicinal diols. They exhibit high enantioselectivities for their substrates, and can be effectively used in the resolution of racemic epoxides prepared by chemical means. As shown in FIG. 1, the selective hydrolysis of a racemic epoxide can generate both the corresponding diols and the unreacted epoxides with high enantiomeric excess (ee) values. However, in order to fully realize the potential of EHs in industrial applications, the following significant limitations urgently need to be overcome: (1) the number of enzymes available is small; and (2) the scope of substrates is limited.
Among the available enzymes, many have selectivity for only one enantiomer limiting access to both enantiomers of a particular target. High concentrations of enzymes and low substrate concentration are required in current synthetic applications because of low catalytic efficiency particularly at high substrate/product concentrations.
As mentioned above, there is currently a need in the biotechnology and chemical industry for molecules that can optimally carry out biological or chemical processes (e.g., enzymes). For example, molecules and compounds that are utilized in both established and emerging chemical, pharmaceutical, textile, food and feed, and detergent markets must meet stringent economical and environmental standards. Expensive processes, which produce harmful byproducts and which suffer from poor or inefficient catalysis, often hamper the synthesis of polymers, pharmaceuticals, natural products and agrochemicals. Enzymes, for example, have a number of remarkable advantages, which can overcome these problems in catalysis: they act on single functional groups, they distinguish between similar functional groups on a single molecule, and they distinguish between enantiomers. Moreover, they are biodegradable and function at very low mole fractions in reaction mixtures. Because of their chemo-, regio- and stereospecificity, enzymes present a unique opportunity to optimally achieve desired selective transformations. These are often extremely difficult to duplicate chemically, especially in single-step reactions. The elimination of the need for protection groups, selectivity, the ability to carry out multi-step transformations in a single reaction vessel, along with the concomitant reduction in environmental burden, has led to the increased demand for enzymes in chemical and pharmaceutical industries.
Enzyme-based processes have been gradually replacing many conventional chemical-based methods. A current limitation to more widespread industrial use is primarily due to the relatively small number of commercially available enzymes. Only ˜300 enzymes (excluding DNA modifying enzymes) are at present commercially available from the >3000 non DNA-modifying enzyme activities thus far described.
The use of enzymes for technological applications also may require performance under demanding industrial conditions. This includes activities in environments or on substrates for which the currently known arsenal of enzymes was not evolutionarily selected. However, the natural environment provides extreme conditions including, for example, extremes in temperature and pH. A number of organisms have adapted to these conditions due in part to selection for polypeptides than can withstand these extremes.
Enzymes have evolved by selective pressure to perform very specific biological functions within the milieu of a living organism, under conditions of temperature, pH and salt concentration. For the most part, the non-DNA modifying enzyme activities thus far identified have been isolated from mesophilic organisms, which represent a very small fraction of the available phylogenetic diversity. The dynamic field of biocatalysis takes on a new dimension with the help of enzymes isolated from microorganisms that thrive in extreme environments. For example, such enzymes must function at temperatures above 100° C. in terrestrial hot springs and deep sea thermal vents, at temperatures below 0° C. in arctic waters, in the saturated salt environment of the Dead Sea, at pH values around 0 in coal deposits and geothermal sulfur-rich springs, or at pH values greater than 11 in sewage sludge. Environmental samples obtained, for example, from extreme conditions containing organisms, polynucleotides or polypeptides (e.g., enzymes) open a new field in biocatalysis. By rapidly screening for polynucleotides encoding polypeptides of interest, the invention provides not only a source of materials for the development of biologics, therapeutics, and enzymes for industrial applications, but also provides a new materials for further processing by, for example, directed evolution and mutagenesis to develop molecules or polypeptides modified for particular activity, specificity or conditions.
In addition to the need for new enzymes for industrial use, there has been a dramatic increase in the need for bioactive compounds with novel activities. This demand has arisen largely from changes in worldwide demographics coupled with the clear and increasing trend in the number of pathogenic organisms that are resistant to currently available antibiotics. For example, while there has been a surge in demand for antibacterial drugs in emerging nations with young populations, countries with aging populations, such as the U.S., require a growing repertoire of drugs against cancer, diabetes, arthritis and other debilitating conditions. The death rate from infectious diseases has increased 58% between 1980 and 1992 and it has been estimated that the emergence of antibiotic resistant microbes has added in excess of $30 billion annually to the cost of health care in the U.S. alone. (Adams et al., Chemical and Engineering News, 1995; Amann et al, Microbiological Reviews, 59, 1995). As a response to this trend pharmaceutical companies have significantly increased their screening of microbial diversity for compounds with unique activities or specificity. Accordingly, the invention can be used to obtain and identify polynucleotides and related sequence specific information from, for example, infectious microorganisms present in the environment such as, for example, in the gut of various macroorganisms.
Identifying novel enzymes in an environmental sample is one solution to this problem. By rapidly identifying polypeptides having an activity of interest and polynucleotides encoding the polypeptide of interest the invention provides methods, compositions and sources for the development of biologics, diagnostics, therapeutics, and compositions for industrial applications.
Chiral epoxides and diols are key building blocks for the synthesis of pharmaceuticals. The epoxide group is readily transformed into a wide range of derivatives by acid or base-catalyzed ring opening reactions, while the diols similarly can be converted into a diverse range of structures. Epoxides have broad applications in areas such as anticancer agents, beta-blockers, beta agonists, antivirals, antifungals, and antibacterials. Opportunities for chiral epoxides exist in both the small synthon area, including C-3 and C-4 units, and the advanced chemical intermediate area for pharmaceuticals.
The C-3 synthons are of major significance because they are used in the processes of many pharmaceuticals and can also lead to a wide range of downstream products. Glycidols (S-(1), and R-(2)) are the leading chiral epoxides among representative C-3 synthons shown in FIG. 2. For example, R-glycidol is used as a building block for atenolol (an antihypertensive drug) and S-glycidol leads to R-glycidyl butyrate (7), an important synthon in the synthesis of oxazolidinone antibiotics. Oxazolidinones represent a relatively new class of antibiotics and currently there are over 40 at various stages of clinical development. There is also an increasing demand for both R- and S-epichlorohydrin (3, 4). Among C-4 synthons, 3,4-epoxy-1-butene (8) is a small molecule with vast potential for the chemical industry. Epoxide 8 leads to the production of over 30 other chiral epoxides that are not readily available. Epoxide 10 is used in the production of saquinavir, an antiviral drug, while its diastereoisomer 11 is used in the synthesis of amprenavir, another antiviral drug (FIG. 3). The mixture of the two compounds can be prepared from phenylalanine through an alkene intermediate. Another epoxide, 12, is the building block for the synthesis of two anticancer drugs, docetaxel and paclitaxel (FIG. 4).
Chemical Asymmetric Synthesis of Epoxides and Diols
Currently available chemical methods for the asymmetric epoxidation of alkenes are the Sharpless asymmetrical epoxidation, the Jacobsen epoxidation, and the method developed by Yian Shi. The Sharpless method uses titanium-based catalysts to epoxidize a wide variety of allylic alcohols with optical yields often greater than 90%. (Johnson, R. A.; Sharpless, K. B. Catalytic asymmetric epoxidation of allylic alcohols. In Catalytic Asymmetric Synthesis; Ojima, I. Ed.; VCH: New York, 1993; pp. 103-158.) This methodology is compatible with a wide range of functionalities and this has led to its extensive use in synthetic chemistry. However, the Sharpless approach suffers a significant drawback as the alkenes must have hydroxyl functionality in the allylic position. In contrast to the Sharpless reaction, the asymmetric epoxidation methodology developed by Jacobsen and Katsuki, ** (Jacobsen, E. N. Asymmetric catalytic epoxidation of unfunctionalized olefins. In Catalytic Asymmetric Synthesis; Ojima, I. Ed.; VCH: New York, 1993; pp. 159-202; and Katsuki, T. Coord. Chem. Rev. 1995, 140, 189-214) which uses optically active (salen)manganese(III) complexes, does not require allylic alcohols. However, the scope of the reaction is somewhat limited due to the steric and electronic nature of the catalysts and the best substrates are cis-alkenes conjugated with aryl, acetylenic and alkenyl groups. This substrate requirement greatly limits the applicability of this method as well. Shi Yan's asymmetric epoxidation method, which uses oxiranes derived from oxone and chiral ketones, is effective for trans- and disubstituted olefins. (Zhi-Xian Wang et al., “An Efficient Catalytic Asymmetric Epoxidation Method,” J. Am. Chem. Soc. 1997, 119, 11224-11235.) However, the use of oxone and the catalytic efficiency are two barriers that hamper its industrial application.
In the case where diols are the desired product, an alternative to epoxidation followed by hydrolysis, is the direct asymmetric dihydroxylation of alkenes. The most successful method for catalytic asymmetric dihydroxylation (AD) of alkenes to generate vicinal diols was developed by Sharpless. (Johnson, R. A.; Sharpless, K. B. Catalytic asymmetric dihydroxylation. In Catalytic Asymmetric Synthesis; Ojima, I. Ed.; VCH: New York, 1993; pp. 227-272.) This uses osmium-based catalysts and is applicable to a wide range of alkenes. The method, however, is not effective for some cis-alkenes. More importantly, the use of osmium which is very toxic prohibits its use for pharmaceutical production.
A different strategy of preparing chiral epoxides and diols is via hydrolytic kinetic resolution of racemic epoxides. The method currently used in industry, based on the (salen)cobalt catalysts developed by Jacobsen, is quite efficient on terminal epoxides. (Tokunaga, M.; Larrow, J. F.; Kakiuchi, F.; Jacobsen, E. N. Science 1997, 277, 936.) However, it is ineffective for the internal epoxides. In addition, it is not applicable for many heteroatom-containing substrates (e.g., pyridyl-type epoxides) due to interference of these atoms with the metal catalysts.
All of the methods discussed above are limited in their application to process scale chiral synthesis by problematic features that include the use of expensive metal catalysts, low substrate/catalyst ratios, and limited efficiency and productivity with varying degrees of enantioselectivities. To overcome these obstacles, attention has turned to biocatalysts. (Besse, Pl ; Veschambre, H. Tetrahedron. 1994, 50, 8885-8927.) Direct stereospecific epoxidation of alkenes by monooxygenases (e.g. cytochromes P450s or other monooxygenases) has been reported. (Archelas, A.; Furstoss, R. Top. Curr. Chem. 1999, 200, 159-191.) These enzyme-catalyzed reactions often give high enantiomeric excesses, but with low yields. Epoxides may be produced indirectly from alkenes by haloperoxidases, via initial halohydrin formation and subsequent ring closure. (Besse, Pl; Veschambre, H. Tetrahedron. 1994, 50, 8885-8927.) Although these enzymes possess great potential for use in the synthesis of enantiopure epoxides, there are also severe limitations for their industrial applications as they all require cofactors, have complex, multi-component structures and generally are not very stable. These limitations pose significant challenges for both the discovery of these enzymes and the development of large-scale industrial biocatalytic applications.
The clear potential demonstrated by the microbial EHs has prompted researchers to explore their use in preparative scale synthesis of epoxides and diols. Shown in Scheme 8 are representative examples in which multi-grams of epoxides and/or diols were made with high ee values. (Choi, et al., Appl. Microbiol. Biotechnol. 1999, 53, 7-11; Guerard, et al., J. Eur. J. Org. Chem. 1999, 3399-3402; Goswami, et al., Tetrahedron: Asymmetry 1999, 10, 3167-3175; Cleij, M.; Archelas, A.; Furstoss, R. Tetrahedron: Asymmetry 1998, 9, 1839-1842; and Genzel, Y.; Archelas, A.; Broxterman, Q. B.; Furstoss, R. Tetrahedron: Asymmetry 2000, 11, 3041-3044.) However, several obstacles must be overcome before a broad industrial platform for EH catalyzed synthesis of epoxides and diols can be realized. First, the number of enzymes available is still small and those that have shown promise in synthetic applications are even more rare. Current discovery of new EHs through screening available strains is hampered by limited culture collections and the lack of powerful screening assays. Secondly, the available enzymes have limited substrate scope and are selective for only one enantiomer as their substrate. For example, A. niger EH prefers styrene-oxide types of substrates, and hydrolyzes R-enantiomers in all the transformations in FIG. 5. Lastly, in most of these preparations, high concentrations of enzymes (either whole cells or crude extract) and rather low substrate concentrations had to be used because of the enzymes' low catalytic efficiency.
Novel EHs need to be discovered to offer complementary enantioselectivity (for example, those that recognize S-enantiomers). EHs suitable for large-scale preparation of different types of epoxides also need to be discovered. Equally important is to improve the stereoselectivity and activity of the existing and new EHs using protein engineering technologies.